The use of Escherichia coli bacteria as the host in molecular biology is well known. This microbe apparently was the first prokaryote used for the commercial production of insulin via a cloned gene. E. coli as a host for a eukaryotic gene encoding a useful polypeptide or protein is not without defects. One defect which received early extensive notoriety is the production of an endotoxin by E. coli. The presence of an endotoxin in a preparation of protein product is not desirable. Thus, extensive work has been done to alleviate this problem. Another defect with use of E. coli as a host is that the microbe produces proteases which can destroy susceptible protein product produced by the microbe. This destruction of protein product could lead to the wrong conclusion of low levels of expression of some cloned eukaryotic genes. Generally, the proteases act upon incomplete or abnormal proteins produced by the microbe. However, it is known that E. coli proteases also act adversely upon useful foreign proteins, for example, human beta preinterferon and somatostatin.
The publication "Stabilization of proteins by a bacteriophage T4 gene cloned in Escherichia coli" by L. D. Simon et al., Proc. Natl. Acad. Sci. USA, Vol 80, pp. 2059-2062, April 1983, discloses the use of a cloned T4 pin gene to stabilize three different kinds of proteins in E. coli cells, i.e., (1) incomplete proteins, e.g., puromycyl polypeptides, (2) abnormal but complete proteins, e.g., the .lambda. tsO protein, and (3) labile eukaryotic proteins encoded by genes cloned in E. coli , e.g., mature human fibroblast interferon. The process described in this publication involves the use of two apparently hybrid plasmids transformed into the E. coli host. One plasmid contains the T4 pin gene whereas the other contains the eukaryotic gene encoding a desired protein product. Though this process allegedly stabilizes the indicated proteins, it is not a desirable commercial process because of the use of two different plasmids in the E. coli host. The compatability of such plasmids may not be as stable as desired in the commercial production of useful proteins by recombinant means. Alteration or loss of compatability can wreak havoc on a commercial operation. The process of the subject invention avoids the issue of plasmid incompatability by use of a doubly chimeric plasmid from which a foreign DNA sequence is recombined into a T-even bacteriophage, which is then used to infect the E. coli host. The stabilization aspect of the subject invention is not based on the cloning of the T4 pin gene as is the case with the above-disclosed publication process. There are several other publications which concern the field of this invention. Complete citations to these publications are found in the "References" section following the examples.
(1) Revel and Georgopoulous, 1969. Disclosure of phage and bacterial mutants which allow the synthesis of phage DNA with HMC (5-hydroxymethyl cytosine). PA0 (2) Snyder et al., 1976. Same as (1) above except cytosine is used in place of HMC. PA0 (3) Kaplan and Nierlich, 1975. Disclosure that EcoRI functions inefficiently on T4 DNA containing HMC. PA0 (4) Shub and Casna, 1981. Abstract of method for inserting fragments of DNA into bacteriophage T4. PA0 (5) Casna and Shub, 1982. Publication of the cloning procedure disclosed herein. Thus, this publication is hereby incorporated herein by reference thereto. PA0 (1) construction of a chimeric plasmid comprising parts of the rII genes of bacteriophage T4 (DNA fragment) and a portion of the genome of an E. coli plasmid, said DNA fragment having sufficient homology to recombine to bacteriophage T4: PA0 (2) in vitro insertion of foreign DNA into the T4 sequence of the chimeric plasmid to give a doubly chimeric plasmid, wherein said DNA fragment from bacteriophage T4 is flanking each end of said foreign DNA; PA0 (3) in vivo recombination between bacteriophage T4 and the doubly chimeric plasmid which transfers the foreign DNA to bacteriophage T4; and, PA0 (4) infection of the E. coli host with T4 harboring the doubly chimeric plasmid. PA0 E. coli 3000X111--ECGSC 5263 PA0 E. coli C600--ATCC 23724 PA0 E. coli 1078--NRRL B-15476. (This NRRL deposit